Anticancer potential of diarylidenyl piperidone derivatives,HO-4200 and H-4318, in cisplatin resistant primary ovarian cancer

We have previously developed a novel class of bi-functional compounds based on a diarylidenyl-piperidone (DAP) backbone conjugated to an N-hydroxypyrroline (-NOH; a nitroxide precursor) group capable of selectively inhibiting STAT3 activation, translocation, and DNA binding activity. HO-4200 and H-4318 are 2 such derivatives capable of inducing apoptosis in ovarian cancer cells through this mechanism and demonstrated efficacy in platinum resistant primary ovarian cancer cell populations and tumor tissues. The improved absorption and cellular uptake of HO-4200 by cancer cells was determined using optical and electron paramagnetic resonance spectrometry. Treatment of ovarian cancer cells with HO-4200 and H-4318 resulted in cleavage of caspase proteins 3, 7, and 9, as well as PARP and inhibition of the pro-survival protein, Bcl-xL, resulting in significantly decreased cell survival and increased apoptosis. HO-4200 and H-4318 significantly inhibit fatty acid synthase (FAS) and pSTAT3 and decreased the expression of STAT3 target proteins: Survivin, c-myc, Bcl-xl, Bcl-2, cyclin D1/D2, and VEGF were suppressed as analyzed using quantitative real time PCR. In addition, HO-4200 and H-4318 significantly inhibited migration/invasion, in primary ovarian cancer cell populations isolated from primary and recurrent ovarian cancer patients. Treatment of freshly collected human ovarian tumor sections with HO-4200 demonstrated significant suppression of pSTAT3 Tyr 705, angiogenesis (VEFG), and markers of proliferation (Ki-67) in ex vivo models. We have shown, for the first time, that the DAP compounds, HO-4200 and H-4318, inhibit cell migration/invasion and induce apoptosis by targeting FAS/STAT3 in human ovarian cancer cells, including primary ovarian cancer cell populations and tumor tissues. Therefore, our results highlight the clinical anti-cancer potential of HO-4200 and H-4318.     

Serine 727 phosphorylation of STAT3: An early change in mouse hepatocarcinogenesis induced by neonatal treatment with diethylnitrosamine

STAT3 activation is involved in development and progression of hepatocellular carcinoma (HCC). We investigated STAT3 activation during hepatocarcinogenesis induced by neonatal diethylnitrosamine (DEN) treatment in mice. Nuclear accumulation and phosphorylation of STAT3 were detected in altered hepatocyte foci in the early stages as well as adenomas and HCCs in the late stages. Although total STAT3 levels were the same between the hepatic lesions and normal livers, S727‐phosphorylated STAT3 was enhanced in adenomas and HCCs, whereas Y705‐phosphorylated STAT3 was detected mainly in HCCs. In mouse HCC cell lines, although both S727 and Y705 remained un‐ or hypophosphorylated under serum‐free conditions, fetal bovine serum (FBS) induced strong S727/weak Y705 phosphorylation, STAT3 nuclear accumulation and cell proliferation, whereas IL‐6 treatment without FBS caused Y705 phosphorylation without S727 phosphorylation, STAT3 nuclear accumulation or cell proliferation. When HCCs were simultaneously treated with FBS/IL‐6, selective suppression of S727 phosphorylation by an MEK inhibitor prevented STAT3 nuclear accumulation and cell proliferation. Furthermore, an S727 phosphorylation‐deficient STAT3 mutant (S727A) had a diminished capacity to accumulate in the nucleus when compared with wild‐type (WT) or the phosphorylation‐mimic mutant (S727D) following treatment with FBS/IL‐6. After treatment with FBS/IL‐6, the cells expressing the S727A mutant proliferated more slowly than those expressing WT or S727D mutant. In contrast, suppression of Y705 phosphorylation by a JAK inhibitor in the FBS/IL‐6 treated cells did not affect STAT3 nuclear accumulation or cell proliferation. Taken together, these data demonstrate that STAT3 activation, mainly through S727 phosphorylation, contributes to the DEN‐induced hepatocarcinogenesis at the earliest stages. © 2013 Wiley Periodicals, Inc.     

Betulinic acid suppresses STAT3 activation pathway through induction of protein tyrosine phosphatase SHP‐1 in human multiple myeloma cells

STAT3 activation has been associated with survival, proliferation and invasion of various human cancers. Whether betulinic acid, a pentacyclic triterpene, can modulate the STAT3 pathway, was investigated in human multiple myeloma (MM) cells. We found that betulinic acid inhibited constitutive activation of STAT3, Src kinase, JAK1 and JAK2. Pervanadate reversed the betulinic acid‐induced downregulation of STAT3 activation, suggesting the involvement of a protein tyrosine phosphatase (PTP). Furthermore, betulinic acid induced the expression of the PTP SHP‐1 and silencing of the SHP‐1 gene abolished the ability of betulinic acid to inhibit STAT3 activation and rescued betulinic acid‐induced cell death. Betulinic acid also downregulated the expression of STAT3‐regulated gene products such as bcl‐xL, bcl‐2, cyclin D1 and survivin. This correlated with an increase in apoptosis as indicated by an increase in the sub‐G1 cell population and an increase in caspase‐3‐induced PARP cleavage. Consistent with these results, overexpression of constitutive active STAT3 significantly reduced the betulinic acid‐induced apoptosis. Betulinic acid also enhanced the apoptosis induced by thalidomide (from 10 to 55%) and bortezomib (from 5 to 70%) in MM cells. Overall, our results suggest that betulinic acid downregulates STAT3 activation through upregulation of SHP‐1, and this may have potential in sensitization of STAT3 overexpressing tumors to chemotherapeutic agents.     

Overexpression of Bcl-2 induces STAT-3 activation via an increase in mitochondrial superoxide

We recently reported a novel interaction between Bcl-2 and Rac1 and linked that to the ability of Bcl-2 to induce a pro-oxidant state in cancer cells. To gain further insight into the functional relevance of this interaction, we utilized computer simulation based on the protein pathway dynamic network created by Cellworks Group Inc. STAT3 was identified among targets that positively correlated with Rac1 and/or Bcl-2 expression levels. Validating this, the activation level of STAT3, as marked by p-Tyr705, particularly in the mitochondria, was significantly higher in Bcl-2-overexpressing cancer cells. Bcl-2-induced STAT3 activation was a function of GTP-loaded Rac1 and NADPH oxidase (Nox)-dependent increase in intracellular superoxide (O₂^•−). Furthermore, ABT199, a BH-3 specific inhibitor of Bcl-2, as well as silencing of Bcl-2 blocked STAT3 phosphorylation. Interestingly, while inhibiting intracellular O₂^•− blocked STAT3 phosphorylation, transient overexpression of wild type STAT3 resulted in a significant increase in mitochondrial O₂^•− production, which was rescued by the functional mutants of STAT3 (Y705F). Notably, a strong correlation between the expression and/or phosphorylation of STAT3 and Bcl-2 was observed in primary tissues derived from patients with different sub-sets of B cell lymphoma. These data demonstrate the presence of a functional crosstalk between Bcl-2, Rac1 and activated STAT3 in promoting a permissive redox milieu for cell survival. Results also highlight the potential utility of a signature involving Bcl-2 overexpression, Rac1 activation and STAT3 phosphorylation for stratifying clinical lymphomas based on disease severity and chemoresistance.     

MicroRNA regulation and therapeutic targeting of survivin in cancer

Survivin, the smallest member of IAP (inhibitor of apoptosis) family, is a dual functional protein acting as a critical apoptosis inhibitor and key cell cycle regulator. Survivin is usually expressed in embryonic tissues during development and undetectable in most terminally differentiated tissues. Numerous studies demonstrate that survivin is selectively upregulated in almost all types of human malignancies and its overexpression positively correlates with poor prognosis, tumor recurrence, and therapeutic resistance. This differential expression of survivin in tumors and normal tissues draws a great interest to develop survivin-targeted therapy for cancer treatment. Nonetheless, the molecular mechanisms controlling survivin expression in malignant tumor cells have not been fully understood. While aberrant activation of receptor tyrosine kinases (RTKs) and the downstream signaling, such as PI-3K/Akt, MEK/MAPK, mTOR, and STAT pathways, have frequently been shown to upregulate survivin, recent data suggest that a class of noncoding RNAs, microRNAs (miRNAs) also play an important role in survivin dysregulation in human cancers. Here, we focus on survivin expression-regulated by specific miRNAs binding to the 3’-UTR of survivin mRNA, and summarize the latest advances on survivin-targeted therapy in clinical trials and the therapeutic potential of survivin-targeting miRNAs in cancer.     

HO-3867, a STAT3 inhibitor induces apoptosis by inactivation of STAT3 activity in BRCA1-mutated ovarian cancer cells

BRCA1 plays an important role in DNA damage and repair, homologous recombination, cell-cycle regulation and apoptosis. BRCA-mutated ovarian cancer often presents at an advanced stage, however, tend to have better response to platinum-based chemotherapy as compared with sporadic cases of epithelial ovarian cancer (EOC). In spite of this, most patients will develop a recurrence and eventually succumb to the disease. Preclinical studies are currently investigating natural compounds and their analogs for tumor-directed targets in ovarian cancer. The aim of this study is to investigate whether the STAT3 inhibitor HO-3867, a novel curcumin analog, has a therapeutic effect on BRCA1-mutated ovarian cancer. Our novel agent, HO-3867 and a commercial STAT3 inhibitor, STATTIC, significantly inhibited BRCA-mutated ovarian cancer cells in vitro in a dose- and time-dependent manner. BRCA-mutated ovarian cancer cells treated with HO-3867 exhibited a significant degree of apoptosis with elevated levels of cleaved caspase-3, caspase-7 and PARP. HO-3867 treatment induced more reactive oxygen species (ROS) in BRCA-mutated cells compared with wild-type cells, however, there was no increased ROS when benign ovarian surface epithelial cells were treated with HO-3867. BRCA1-mutated cancer cells had higher expression of Tyrosine-phosphorylated STAT3 (pTyr705) as compared with other STAT proteins. Furthermore, treatment of these cells with HO-3867 resulted in decreased expression of pTyr705 and its downstream targets cyclin D1, Bcl-2 and survivin. In addition, overexpression of STAT3 cDNA provided resistance to HO-3867-induced apoptosis. Our results show that HO-3867, a potent STAT3 inhibitor, may have a role as a biologically targeted agent for BRCA1-mutated cancers either as an adjunct to cytotoxic chemotherapy or as a single agent.